During infections by pathogens, such as viruses, bacteria, and parasites, an important component of the immune defense is provided by natural killer (NK) cells. NK cells are a group of white blood cells (lymphocytes) that can directly kill infected cells and tumor cells. NK cells differ from B and T lymphocytes in that they respond much faster to infections. Unlike B and T cells, NK cells do not express antigen-specific receptors, which raises the interesting question of how NK cells can detect infected cells. NK cells exert their function in two ways: by producing cytokines, such as interferon-gamma, and by killing infected cells. NK cells can also kill tumor cells. Despite the importance of NK cells in the innate immune response to many types of pathogens, it is still unclear what receptors and what signal transduction pathways control their activation. NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex class I (MHC class I) prevent the killing of normal, healthy cells. [unreadable] [unreadable] The major goal of this project is to define the receptor-ligand interactions that regulate cytotoxicity and cytokine production by NK cells, and to determine the precise contribution of individual receptors to signal transduction in NK cells. Both activating and inhibitory receptors are encompassed in our studies. Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. Using Drosophila insect cells, engineered to express defined ligands of human NK cell receptors, we have shown that target cell lysis by NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. Engagement of the adhesion receptor LFA-1 was sufficient to induce polarization of granules, but not degranulation, in NK cells. Conversely, engagement of the low affinity receptor for IgG Fc (CD16) induced degranulation without specific polarization. Lysis by NK cells occurred when polarization and degranulation were induced by the combined presence of ligands for LFA-1 and CD16 on insect cells. This work showed that cytotoxicity by resting NK cells is tightly controlled by separate or co-operative signals from different receptors for granule polarization and degranulation.[unreadable] [unreadable] Certain patients with a genetic defect in a molecule called syntaxin 11 are characterized by a sepsis-like illness with cytopenia, hepatosplenomegaly and deficient lymphocyte cytotoxicity. This fatal disease is known as Familial Hemophagocytic Lymphohistiocytosis 4 (FHL4). Using very limited amounts of blood samples from such patients, we have been able to show that syntaxin 11 is expressed in NK cells and is essential for the release of cytotoxic granules. Interestingly, IL-2stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared to FHL2 and FHL3 patients. Since the effector T cell compartment is still immature in infants, our data suggest that the observed defect in NK cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.[unreadable] [unreadable] [unreadable] Activation of NK cells by contact with target cells is controlled by inhibitory receptors for MHC class I, which include the human killer cell Ig-like receptors (KIR) specific for HLA-C. Inhibitory KIR accumulates at the site of contact with HLA-Cexpressing target cells, and prevents phosphorylation of the guanine exchange factor Vav1. Since Vav1 is known to control polymerization of actin filaments, it is possible that the main target for inhibition by KIR is actin cytoskeleton rearrangements, which are necessary for the formation of a tight interface between NK cells and their target cells, in a so-called immune synapse. Experiments were designed to test whether granule polarization, degranulation, or both were sensitive to inhibition by KIR. A large number of human NK cell clones with defined expression of inhibitory KIR was used and tested with several different types of target cells. Inhibition of degranulation occurred only when NK cells were in contact with one of the human cell lines tested. Whereas degranulation was inhibited only in the context of certain receptor ligand interactions, polarization was inhibited in all cases. Therefore, granule polarization is preferred over degranulation as a target of inhibition.